CN108840848A - 一种香豆素半抗原与抗原的制备方法及应用 - Google Patents
一种香豆素半抗原与抗原的制备方法及应用 Download PDFInfo
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/12—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 3 and unsubstituted in position 7
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
一种香豆素半抗原与抗原的制备方法及应用，其特征在于：所述香豆素半抗原是由香豆素进行缩醛反应得到的；所述香豆素抗原是由香豆素半抗原与载体蛋白偶联得到。本发明制备的抗原呈现出特异性的香豆素抗原决定簇，通过免疫动物可制备香豆素单克隆抗体。产生的抗体特异性高、灵敏度高，可用于制备检测香豆素的酶联免疫试剂盒、胶体金试纸条和时间分辨荧光免疫层析试纸条，从而实现烟草、香精香料及食品中香豆素的快速检测。
Description
技术领域
本发明涉及一种香豆素半抗原与抗原的制备方法及应用。属于免疫化学技术领域。
背景技术
香豆素系化合物是一类具有芳香气味，母核为苯并α-吡喃酮结构的天然产物，广泛分布于植物界，根据结构可分为简单香豆素系化合物、呋喃香豆素系化合物、吡喃香豆素系化合物和其他香豆素系化合物等。香豆素（coumarin）又名邻羟基桂酸内酯和1,2-苯并吡喃酮，是一种天然植物次生产品，是广泛存在于自然界中的一种内酯类化合物，于1820年发现于黑香豆中，主要以游离状态和苷的形式存在于植物中，在芸香科和伞形科植物中最多，其次存在于豆科、兰科、木樨科、茄科和菊科植物，少数来自微生物。香豆素作为一种重要的香料，常用作定香剂、脱臭剂，配制香水和香料，也用作饮料、食品、卷烟、塑料制品、橡胶制品等的增香剂，医药上将其加入药剂中作为矫味剂。实验发现，香豆素对小鼠胚胎有毒性，能引起痛觉消失，使中性胆碱酯酶发生变化，对大鼠为可疑致肿瘤物。香豆素在体内的含量达到一定量时具有生理毒性，通过长期的动物实验发现其具有肝毒性，主要是由代谢的途径导致P450（细胞色素P）及其依赖性酶的减少引起的。美国食品药品管理局（FDA）早在1954年就禁止将香豆素用作食品添加剂；欧洲食品安全局（EFSA）规定香豆素的每日耐受摄入量（TDI）为0.1 mg/kg bw；欧盟规定，对于洗去型化妆品，香豆素的含量不得超过30 μg/g，对于非洗去型化妆品，香豆素含量不得高于10 μg/g；2010年7月，台湾地区食品管理机构发布通知禁止在食品中添加香豆素，同时规定“惟饮料中因使用天然香料，导致天然香料本身含香豆素残留时，其含量仍应在2.0 mg/kg以下”。
目前，香豆素主要通过直接萃取法、超声萃取法、同时蒸馏萃取法、水蒸气蒸馏法等提取，结合气相色谱法（GC）、气相色谱-质谱法（GC-MS）、气相色谱-串联质谱法（GC-MS/MS）、高效液相色谱法（HPLC）、超高效液相色谱-串联质谱法（UPLC-MS/MS）检测。但是由于这些分析方法需要昂贵的大型仪器设备和专业的检测人员、前处理过程复杂、操作繁琐、检测成本高、分析速度慢，难以满足现场监测和大量样本中香豆素含量快速筛查的需要。基于抗原抗体特异性识别的免疫分析方法可以定性定量检测样品中的香豆素含量。这种分析方法对仪器设备要求不高、快速简便，一般无需对样品进行复杂的预处理，灵敏度高、特异性强，对使用人员的专业技术要求不高，容易普及和推广，可满足快速分析检测的需要，尤其适宜现场筛选和大量样品的快速分析。免疫分析为香豆素研究提供了一个新的分析检测途径。免疫分析目前已成为分析研究的一个崭新领域，美国化学会将免疫分析与气相色谱、液相色谱共同列为分析的三大支柱技术。本发明属于小分子化合物免疫化学和残留分析技术领域，涉及有机合成、免疫化学及生物化学等，依靠免疫学、免疫化学基本原理和生物技术手段，设计、合成小分子目标分析物半抗原，并与载体蛋白质偶联，制备有效人工抗原。制备的抗原可以通过免疫动物制备对小分子分析物特异性识别的抗体，利用抗原抗体的特异性免疫学反应和易被检测识别的标记物的放大作用，定量的检测样品中超微量小分子目标物。半抗原的分子设计与合成是产生特异性抗体和建立免疫分析方法的关键步骤。人工抗原的制备，包括结合位点、结合方式、载体种类及半抗原与目标分析物质任何结构上的差异，诸如分子大小、形状、成分、构型、构象、极性、电子云密度等在内的拓扑性征，都可能极大的影响着相应抗体的性质。目前关于香豆素半抗原及抗原的制备方法尚未见报道。
发明内容
本发明的目的正是基于上述现有技术状况而提供一种香豆素半抗原与抗原的制备方法及其应用。
本发明的目的是通过以下技术方案来实现的：
一种香豆素半抗原的制备方法，是由香豆素进行缩醛反应得到的，其分子结构为：
。
具体步骤如下：
1）取香豆素1.00 g，加入80 mL氯苯中搅拌溶解，加2.50 g叔丁醇过氧化氢，搅拌充分混匀，加3-(1,3-二氧杂烷-2-基)丙醛0.89 g，100℃搅拌24 h。停止反应后，加100 mL冷水，用100 mL乙酸乙酯萃取3次，使用50 mL水洗，有机相无水硫酸钠干燥蒸干，60 mL石油醚/二氯甲烷（v/v，1/1）重结晶，得到缩醛香豆素化合物1.70 g。
2）取缩醛香豆素1.70 g，加20 mL乙腈溶解，加1 mol/L稀盐酸10 mL，室温剧烈搅拌4 h，停止反应，旋蒸，除去乙腈，加水50 mL，加1mol/L的NaOH调节pH值到6，加70 mL 1,2-二氯乙烷萃取3次，100 mL水洗，蒸干，上硅胶柱，2 L二氯甲烷/甲醇（v/v，5/1）洗脱，分离纯化，得到丁醛香豆素半抗原1.30 g。
所述香豆素半抗原可用于制作动物免疫的抗原体系原料。
一种香豆素抗原的制备方法，是由所述香豆素半抗原与载体蛋白偶联得到，包括免疫抗原和包被抗原；所述载体蛋白为牛血清白蛋白、卵清蛋白、钥孔血蓝蛋白或人血清白蛋白。
具体步骤如下：
免疫抗原的制备：取香豆素半抗原16 mg，加乙醇溶解，得到A液，取牛血清白蛋白（BSA）0.1 g，加pH值为5.6的醋酸盐缓冲液溶解，得到B液，将A液滴加到B液中，4℃搅拌20 h，加1mg硼氢化钠，继续搅拌2 h，0.02 mol/L PB透析纯化三天，每天换液三次，分装，得到免疫抗原，-20℃保存备用。
包被抗原的制备：取香豆素半抗原5 mg，加乙醇溶解，得到A液，取卵清白蛋白（OVA）50 mg，加pH值为9.1的碳酸盐缓冲液溶解，得到B液，将A液滴加到B液中，4℃搅拌20h，加1 mg硼氢化钠，继续搅拌2 h，0.02 mol/L PB透析纯化三天，每天换液三次，分装，得到包被抗原，-20℃保存，备用。
采用所述香豆素抗原免疫动物得到的单克隆抗体，可用于制备检测香豆素的酶联免疫试剂盒、胶体金试纸条和时间分辨荧光免疫层析试纸条，从而实现烟草及食品中香豆素的快速检测。
本发明中合成的香豆素半抗原既最大程度的保留了香豆素的化学结构，又有合适长度的连接臂，用该半抗原制备的抗原呈现出特异性的香豆素抗原决定簇，使得筛选出高特异性的香豆素单克隆抗体成为可能。产生的抗体特异性高、灵敏度高，可用于建立酶联免疫吸附测定方法和胶体金试纸快速测定法，从而实现烟草及食品中香豆素的快速检测。
附图说明
图1：香豆素半抗原合成路线图。
具体实施方式
下面结合具体的实施例来进一步阐述本发明。应理解，这些实施例仅用于说明本发明，而不用来限制本发明的范围。
实施例1 香豆素半抗原的制备
1、香豆素半抗原的合成
取香豆素1.00 g，加入80 mL氯苯中搅拌溶解，加2.50 g叔丁醇过氧化氢，搅拌充分混匀，加3-(1,3-二氧杂烷-2-基)丙醛0.89 g，100℃搅拌24 h。停止反应后，加100 mL冷水，用100 mL乙酸乙酯萃取3次，使用50 mL水洗，有机相无水硫酸钠干燥蒸干，60 mL石油醚/二氯甲烷（v/v，1/1）重结晶，得到缩醛香豆素化合物1.70 g，收率94.00%。
取缩醛香豆素1.70 g，加20 mL乙腈溶解，加1 mol/L稀盐酸10 mL，室温剧烈搅拌4h，停止反应，旋蒸，除去乙腈，加水50 mL，加1mol/L的NaOH调节pH值到6，加70 mL 1,2-二氯乙烷萃取3次，100 mL水洗，蒸干，上硅胶柱，2 L二氯甲烷/甲醇（v/v，5/1）洗脱，分离纯化，得到丁醛香豆素半抗原1.30 g，收率91.55%。
2、香豆素半抗原的鉴定
取上述半抗原经核磁共振氢谱鉴定，1H NMR（CDCl3, 300MHZ）δ: 9.72 (1H, s, CHO),8.45 (1H, s, C=CH), 7.84 (1H, dd, ArH), 7.65 (1H, dd, ArH), 7.42 (2H, dd,ArH), 3.27 (2H, dd, CH2), 2.51(2H, t, CH2)。
图谱中化学位移δ=9.72的为间隔臂上醛基氢的共振吸收峰，δ=3.27、2.51的为间隔臂上亚甲基氢的共振吸收峰，除香豆素原有氢特征吸收峰外，这些峰的存在，证明间隔臂偶联成功，香豆素半抗原结构正确。
实施例2 香豆素抗原的制备
1、香豆素免疫抗原的合成
香豆素半抗原与牛血清白蛋白（BSA）偶联得到免疫抗原。
取香豆素半抗原16 mg，加乙醇溶解，得到A液，取牛血清白蛋白0.1 g，加pH值为5.6的醋酸盐缓冲液溶解，得到B液，将A液滴加到B液中，4℃搅拌20 h，加1 mg硼氢化钠，继续搅拌2 h，0.02 mol/L PB透析纯化三天，每天换液三次，分装，得到免疫原，-20℃保存备用。
2、香豆素包被抗原的合成
香豆素半抗原与卵清蛋白（OVA）偶联得到包被抗原。
取香豆素半抗原5 mg，加乙醇溶解，得到A液，取卵清蛋白50 mg，加pH 值为9.1的碳酸盐缓冲液溶解，得到B液，将A液滴加到B液中，4℃搅拌20 h，加1 mg硼氢化钠，继续搅拌2 h，0.02 mol/L PB透析纯化三天，每天换液三次，分装，得到包被原，-20℃保存，备用。
3、香豆素抗原的鉴定
按合成香豆素偶联抗原反应所用半抗原、载体蛋白与偶联产物的比例，进行紫外（200~ 400 nm）扫描测定，通过比较三者分别在260 nm和280 nm的吸光度值计算其结合比。偶联物香豆素半抗原-载体蛋白的最大吸收峰与香豆素半抗原、载体蛋白的最大吸收峰相比发生了明显的变化，表明香豆素半抗原-载体蛋白的合成是成功的。经计算，半抗原与BSA的结合比为16:1，与OVA的结合比为12:1。
实施例3 香豆素单克隆抗体的制备
1、杂交瘤细胞的获得
1）动物免疫：将香豆素半抗原-BSA偶联物（免疫原）与等量的弗氏完全佐剂充分乳化，皮下注射6周龄的Balb/c小鼠，每只0.2 mL；此后，每两周加强免疫一次，用弗式不完全佐剂代替弗氏完全佐剂，方法和剂量同首次免疫；
2）最后一次加强免疫一周后眼底静脉采血测定香豆素抗体的效价和抑制作用，有抑制且效价达到1:10000以上时进行如下末次免疫：腹腔注射不加任何佐剂的免疫原溶液0.1mL，三天后处死小鼠，取其脾脏淋巴B细胞与骨髓瘤细胞融合；
3）细胞融合7 d后，用间接ELISA法检测抗体，选择抗体分泌阳性孔，用HT培养基扩大培养后，采用有限稀释法接种于96孔培养板内，选择单个细胞集落孔的培养上清做抗体检测，阳性者用同样方法再次克隆，直至克隆后有杂交瘤细胞生长的抗体分泌阳性率为100%，保存阳性细胞株。
2、单克隆抗体的制备
采用体内诱生法，将Balb/c小鼠（8周龄）腹腔注入灭菌石蜡油0.5 mL/只，7天后腹腔注射杂交瘤细胞5×105个/只，7天后采集腹水。用辛酸-饱和硫酸铵法进行纯化，得到香豆素单克隆抗体溶液（-20℃保存）。
3、单克隆抗体效价的测定
用间接竞争 ELISA法测定抗体的效价为1:(200000~500000)。
间接竞争ELISA方法：用香豆素半抗原-OVA偶联物包被酶标板，加入香豆素标准品溶液、香豆素单克隆抗体溶液和辣根过氧化物酶标记的羊抗鼠抗抗体溶液，25℃反应30min，倒出孔内液体，用洗涤液洗涤3~5次，用吸水纸拍干；加入底物显色液，25℃反应15 min后，加入终止液终止反应；测定OD450。
4、单克隆抗体特异性的测定
将BSA和OVA均稀释至与香豆素半抗原-OVA相同浓度进行包被，再加入含抗体腹水，测试其结合显色情况。选择7-乙氧基-4-甲基香豆素、二氢香豆酯、7-羟基香豆素、3,3’-羰基双（7-二乙氨基香豆素）、7-甲氧基香豆素、莨菪亭、6,7-二甲氧基香豆素、7-甲基香豆素、环香豆素、香兰素、黄樟素、突厥烯酮等香豆素的结构类似物，将其等比稀释液替代香豆素稀释液，其他条件相同，做三次平行，测定它们与香豆素单克隆抗体之间的交叉反应率。以50%抑制的香豆素浓度与50%抑制的类似物浓度的百分比为其交叉反应率，反映单克隆抗体的特异性。
实验结果显示，BSA、OVA均不与香豆素单克隆抗体发生反应，说明筛选获得的香豆素单克隆抗体特异性针对连接在载体蛋白上的香豆素抗原决定簇。表1列出了香豆素单克隆抗体与香豆素结构类似物的交叉反应性。从表1可以看出，香豆素单克隆抗体与7-乙氧基-4-甲基香豆素、二氢香豆酯、7-羟基香豆素、3,3’-羰基双（7-二乙氨基香豆素）、7-甲氧基香豆素、莨菪亭、6,7-二甲氧基香豆素、7-甲基香豆素、环香豆素、香兰素、黄樟素、突厥烯酮均无交叉反应。
表1 香豆素单克隆抗体与香豆素类似物的交叉反应率
| 化合物 | 交叉反应率/% |
| 7-乙氧基-4-甲基香豆素 | 4 |
| 二氢香豆酯 | 3.5 |
| 7-羟基香豆素 | 12 |
| 3,3’-羰基双（7-二乙氨基香豆素） | 4.5 |
| 7-甲氧基香豆素 | 8.3 |
| 莨菪亭 | 8 |
| 6,7-二甲氧基香豆素 | 7.2 |
| 7-甲基香豆素 | 6.3 |
| 环香豆素 | 7.5 |
| 香兰素 | 2 |
| 黄樟素 | 1 |
| 突厥烯酮 | 1 |
Claims (8)
1.一种香豆素半抗原的制备方法，其特征在于：是由香豆素进行缩醛反应得到的，其分子结构式为：
。
2.如权利要求1所述的香豆素半抗原的制备方法，其特征在于：该制备方法的具体步骤如下：
1）取香豆素1.00 g，加入80 mL氯苯中搅拌溶解，加2.50 g叔丁醇过氧化氢，搅拌充分混匀，加3-(1,3-二氧杂烷-2-基)丙醛0.89 g，100℃搅拌24 h；停止反应后，加100 mL冷水，用100 mL乙酸乙酯萃取3次，使用50 mL水洗，有机相无水硫酸钠干燥蒸干，60 mL石油醚/二氯甲烷（v/v，1/1）重结晶，得到缩醛香豆素化合物1.70 g；
2）取缩醛香豆素1.70 g，加20 mL乙腈溶解，加1 mol/L稀盐酸10 mL，室温剧烈搅拌4h，停止反应，旋蒸，除去乙腈，加水50 mL，加1mol/L的NaOH调节pH值到6，加70 mL 1,2-二氯乙烷萃取3次，100 mL水洗，蒸干，上硅胶柱，2 L二氯甲烷/甲醇（v/v，5/1）洗脱，分离纯化，得到丁醛香豆素半抗原1.30 g。
3.如权利要求1所述方法制备的香豆素半抗原的应用，其特征在于：所述香豆素半抗原可用于制作动物免疫的抗原体系原料。
4.一种香豆素抗原的制备方法，其特征在于：是由权利要求1制得的香豆素半抗原与载体蛋白偶联得到。
5.如权利要求4所述的香豆素抗原的制备方法，其特征在于：所述载体蛋白为牛血清白蛋白、卵清蛋白、钥孔血蓝蛋白或人血清白蛋白。
6.如权利要求4所述的香豆素抗原的制备方法，其特征在于：具体步骤如下：取香豆素半抗原16 mg，加乙醇溶解，得到A液，取牛血清白蛋白0.1 g，加pH值为5.6的醋酸盐缓冲液溶解，得到B液，将A液滴加到B液中，4℃搅拌20 h，加1 mg硼氢化钠，继续搅拌2 h，0.02mol/L PB透析纯化三天，每天换液三次，得香豆素抗原；分装， -20℃保存备用。
7.如权利要求4所述的香豆素抗原的制备方法，其特征在于：具体步骤如下：取香豆素半抗原5 mg，加乙醇溶解，得到A液，取卵清白蛋白50 mg，加pH值为9.1的碳酸盐缓冲液溶解，得到B液，将A液滴加到B液中，4℃搅拌20 h，加1 mg硼氢化钠，继续搅拌2 h，0.02 mol/LPB透析纯化三天，每天换液三次，得香豆素抗原；分装，-20℃保存备用。
8.如权利要求4所述方法制备的香豆素抗原的应用，其特征在于：采用香豆素抗原免疫动物得到的单克隆抗体，可用于制备检测香豆素的酶联免疫试剂盒、胶体金试纸条和时间分辨荧光免疫层析试纸条，从而实现烟草、香精香料及食品中香豆素的快速检测。
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